Pravastatin is an inhibitor of HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase), a key enzyme in cholesterol biosynthesis. This enzyme significantly reduces plasma cholesterol and lipid levels and is thereby of great pharmacological importance in the therapy of hypercholesterolemia and hyperlipidemia. [Serizawa et al., J. Antibiot., 36: 887-891 (1983); Serizawa et al., J. Antibiot., 36: 918-920 (1983); Serizawa et al., J. Antibiot., 36: 604-607 (1983); Tsujita et al., Biochim. Biophys. Acta, 877: 50-60 (1986); Arai et al., Ann. Rep. Sankyo Res. Lab., 40: 1-38 (1988); and Koga et al., Biochim. Biophys. Acta, 1045: 115-120 (1990).]
Pravastatin is obtained by microbial hydroxylation of sodium ML-236B (compactin), a substance produced by a filamentous fungus, Penicillium citrinum. This hydroxylation can be effected to differing degrees by many different genera of fungi, such as Mucor Rhizopus, Syncephalastrum, Cunninghamella, Mortierella and bacteria such as Nocardia, Actinomadura and Streptomyces as described in various patents. [U.S. Pat. Nos. 5,179,013; 4,448,979; 4,346,227; 4,537,859; 6,566,120; 6,750,366; Canadian patent Nos. 1,150,170; 1,186,647; Japanese Patent No. 58-10572; and European Patent No. 0605230]
The hydroxylation takes place at the 6-position of ML-236B, catalyzed by a Cytochrome P-450sca monooxygenase system, found in Streptomyces carbophilus. [Matsuoka et al., Eur. J. Biochem., 184: 707-713 (1989); and Serizawa, et al., Biochimica et Biophysica Acta, 1084: 35-40 (1991).] Cytochrome P-450sca was characterized as occurring in three forms: P-450sca-1, P-450sca-2, and P-450sca-3, which according to U.S. Pat. No. 5,179,013, are suitable for use in hydroxylation processes.
Serizawa et al. cloned the DNA encoding P-450sca-2 from Streptomyces Carbophilus. [Japanese Patent Kokai No. 6-70780; and Watanabe et al., Gene, 163: 81-85 (1995).] The gene has an open reading frame of 1233 bp, encoding a 410 amino acid protein. [Watanabe et al., Gene, 163: 81-85 (1995).] A 2.8 kb DNA insert, along with a 1 kbp portion of the 5′-noncoding region of the P-450sca-2 gene, was cloned into a multicopy plasmid, pIJ702, and used to transform Streptomyces lividans TK21. The transformed Streptomyces lividans TK21 converted ML-236B to pravastatin even faster than S. carbophilus. See, Watanabe, I. et al., Gene 163:81-85 (1995).
Watanabe et al. disclosed that the expression of P-450sca is subject to substrate induction of transcription, that is, ML-236B and phenobarbital were found to enhance the expression of P-450 by as much as 30-fold. This was established by Northern blotting, which found no transcription in the absence of ML-236B, but which found three transcripts when ML-236B was present. The levels of transcription increased over a period of six hours to a maximum rate when substrate was present. The DNA sequence of the 5′ region was published, which coded a regulator like protein and the promoter sequence. [Watanabe et al., Gene 210, 109-116 (1998).]
A 1 kbp length of the 5′-noncoding region of the gene encoding cytochrome P-450sca-2 in the Streptomyces carbophilus has transcription promoter activity, which is substrate inducible. When the 1 kbp region was shortened, the transcription promoter allowed significant expression of the protein in a suitable expression system without having to be induced. [U.S. Pat. No. 5,830,695.]
Serizawa and Matsuoka purified a NADH-cytochrome-P-450-reductase from S. carbophilus. They used purified P-450sca protein to demonstrate in vitro hydroxylation activity in the presence of the purified flavoprotein (NADH-cytochrome-P-450-reductase), NADH and O2. They disclosed that P-450sca and NADH-cytochrome-P-450 reductase reconstituted the hydroxylation activity in vitro and did not obtain any evidence for existence of iron-sulfur protein in S. carbophilus. Based on these findings, Serizawa and Matsuoka classified the P-450sca cytochrome system as a two component system similar to those found in microsomal cytochrome systems of eukaryotes, which do not require iron-sulfur proteins. [Serizawa and Matsuoka, Biochem et Biophysica Acta, 1084: 35-40 (1991).]
The present invention is based on the discovery that the gene encoding fdxshe or a fdxshe-like protein is located downstream from the cytochrome P-450 gene and is necessary for the function of the P-450sca cytochrome. Thus, the P-450sca cytochrome system is a three component system, in contradistinction to the proposed two component system proposed by Serizawa and Matsuoka, as described in FIG. 4.